Infection with the human parvovirus B19 usually results in a mild disease known as erythema infectiosum, or fifth disease

The zymogen of bacterial transglutaminase was found during cultivation of Streptoverticillium mobaraense (DSMZ strain) using rabbit antibodies raised against the active enzyme. Ion‐exchange chromatography at pH 5.0 yielded a highly purified pro‐enzyme. Structure information was obtained by means of Edman degradation and analysis of PCR amplified nucleotide fragments. The data revealed an excess of negatively charged amino acids in the pro‐region resulting in a decreased isoelectric point of the zymogen. Additionally, the new sequence gave rise to some modifications to the previously published hypothetical structure of prepro‐transglutaminase derived from genomic DNA [Washizu, K., Ando, K., Koikeda, S., Hirose, S., Matsuura, A., Takagi, H., Motoki, M. & Takeuchi, K. (1994) Biosci. Biotechnol. Biochem. 58, 82−87]. Inactive transglutaminase, which carries an activation peptide of 45 amino acids, has a calculated molecular mass of 42 445 Da. Its pro‐region provides for both suppression of activity and increased thermostability. Furthermore, it could be shown that the micro‐organism produces a protease which cleaves pro‐transglutaminase at the C‐side of Pro45. Rapid transformation of the mature enzyme also occurs by addition of other proteases. During conversion, 43 and 41 amino acid peptides are released by bovine trypsin and dispase from Bacillus polymyxa, respectively. The detection of endogenous substrates in the murein layer makes discussion of the physiological role of bacterial transglutaminases necessary.

Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 μg/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 μg/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development.

Objective. To show a possible association between parvovirus B19 infection and the presence of antiphospholipid antibodies (aPL) in patients with rheumatic diseases.Methods. Serum samples obtained from 88 children with various forms of juvenile rheumatic disease and from 40 adults with systemic lupus erythematosus, the antiphospholipid syndrome, or other rheumatic disease, who had previously been tested and shown to be positive for IgG aPL, were analyzed for the presence of B19 DNA, for antibodies against the B19 viral proteins VP1, VP2, and NS1, and for IgG aPL (anticardiolipin, anti-␤ 2 -glycoprotein I, and antiphosphatidylserine). As controls, serum samples obtained from 135 children with noninflammatory bone diseases or growth retardation were also analyzed.Results

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1 Extracellular nucleotides can activate a common purinoceptor mediating various cell responses. In this study we report that stimulation of rat mesangial cells with ATP and UTP leads to a rapid activation of the protein kinase B/Akt (PKB) pathway. Time-course studies reveal a rapid and transient phosphorylation of both Ser 473 and Thr 308 of PKB with a maximal eect after 5 min of stimulation. The response is concentration-dependent with a maximal eect at 30 mM of ATP and UTP. Western blot analysis of mesangial cells reveals the expression of the isoenzymes PKB-a and PKB-g, but not the PKB-b. 2 ATP and UTP also activate the upstream located PI 3-kinase-dependent kinase. Furthermore, the ATP-and UTP-induced PKB phosphorylation is abolished by two inhibitors of the PI 3-kinase. In addition, suramin, a putative P2Y 2 receptor antagonist, and pertussis toxin, an inhibitor of G i /G o activation, markedly block ATP-and UTP-induced PKB phosphorylation. 3 A series of ATP and UTP analogues were tested for their ability to stimulate PKB phosphorylation. UTP, ATP and g-thio-ATP are the only compounds capable of activating PKB. 4 Stress-induced apoptosis of mesangial cells is reduced by the stable ATP analogue, g-thio-ATP, and this inhibitory eect is reversed in the presence of LY 294002. 5 In summary, these results demonstrate that extracellular nucleotides are able to activate the PI 3-kinase/PDK/PKB cascade via the P2Y 2 -receptor and a pertussis toxin-sensitive G i protein.Moreover, in mesangial cells this cascade may have an important role in the antiapoptotic response but not in the mitogenic or in¯ammatory response produced by extracellular nucleotides.

The unique region of structural protein VP1 of parvovirus B19 (erythrovirus B19) is important for eliciting neutralizing antibodies that are responsible for eliminating the virus from the peripheral blood and for inducing lifelong immunity. Neutralizing human MAbs bind a conformationally defined epitope spanning VP1 residues 30-42. The DNA sequence encoding the VP1-unique region was determined in parvovirus B19 isolated from peripheral blood and amniotic fluid of nine acutely infected pregnant women, five arthritis patients and two chronically infected children. The amino acid sequences of the VP1-unique region exhibited higher variability in comparison with other B19-specific proteins. To analyse the influence of amino acid variations on antibody binding and protein conformation, two variants of the VP1-unique region were selected and expressed in E. coli as intein-fusion proteins. The selected variants displayed a number of amino acid exchanges in the VP1-unique region and had mutations in the determined epitope and adjacent regions. After purification via affinity chromatography, the dissociation constants K D of VP1-specific human MAbs interacting with the variant antigens and a viral prototype of the VP1-unique region were determined with a quartz crystal microbalance biosensor. A value of 5n4i10 N8 M was determined for the prototype isolate pJB ; the affinity constants for the variant VP1-unique regions were similar. Comparable values were obtained for interaction of antibodies with non-infectious VP1/VP2 capsids produced by recombinant baculovirus and with B19 virions from amniotic fluid. It is concluded that the conformation of the epitope is unaffected by mutations or the environment of the VP1-unique region in virus capsids.

These data suggest that ceramide represents an important mediator of reactive oxygen and nitrogen species-triggered cell responses, like apoptosis. There seem to be cell type-specific protective mechanisms that critically depend on a fine-tuned redox balance between reactive nitrogen and oxygen species to determine whether a cell undergoes apoptosis or survives when exposed to oxidative and/or nitrosative stress conditions.